Advances in tools and technologies for multiplexed bioluminescence imaging

Bioluminescence is a popular method for noninvasive imaging of cells and other features. Visualizing multiple probes at once, though, has been historically challenging due to limited toolsets and suitable detection methods. This perspective focuses on recent advances in bioluminescence technology that are lowering the barrier to multiplexed imaging. We first discuss new luciferins and luciferases that are capable of resolving multiple targets. We then showcase the latest developments in luciferase-fluorescent probe engineering for multi-color readouts. Finally, we describe innovations in detection hardware for easier capture and resolution of bioluminescent emitters. Together, these advances are pushing the frontiers of what can be observed in living systems.